Project Summary/Abstract The long term goal of this research is to determine the molecular basis of membrane traffic in mammalian cells. The focus is on mannose 6-phosphate receptors (MPRs) that deliver newly synthesized lysosomal enzymes from the Golgi to pre-lysosomes, and then return to the Golgi to pick up more cargo. Several recently discovered proteins are needed for MPR transport from late endosomes to the trans Golgi network: a cargo selection protein that recognizes the MPRs in late endosomes (TIP47), a pathway- specific SNARE complex for fusion of MPR-vesicles at the TGN, and two proteins that function in vesicle tethering at the Golgi (GCC185 and RhoBTB3). The goals of this application are (1) to define precisely, the distinct routes taken by cargoes that are transported from early endosomes back to the Golgi, with focus on MPRs in comparison with cholera toxin; (2) to carry out a genome-wide, automated siRNA screen for proteins needed for MPR recycling. The screen will make use of the fact that depletion of proteins needed for MPR recycling leads to dispersal of MPRs into peripherally localized cellular compartments. Computer software can detect this dispersal, permitting automated analysis of the effects of 22,000 siRNAs transfected into cultured cells. (3) Also proposed are experiments to further characterize two novel Rab9 effectors that are important for this trafficking pathway: RhoBTB3 and RUTBC1. In summary, these experiments open up entirely new areas of investigation in the area of MPR trafficking and will provide fundamental information regarding the mechanisms of receptor trafficking in human cells. The work has broad application to our understanding of a number of disease states including diabetes, cancer, heart disease and neurological disorders.